This project seeks to understand chloride conductance regulatory pathways that have recently been demonstrated to control the osmotically-sensitive chloride current, I/Cl.swell. The rationale for this approach to the treatment of cystic fibrosis is that I/Cl.swell might be used as an alternate conductance pathway to the defective CFTR conductance. Alternatively, the newly defined regulatory proteins may be found to control chloride conductances in addition to I/Cl.swell. If chloride conductances can be regulated, the potential exists for activation of these alternate chloride conductance pathways to replace CFTR function. We have previously characterized the swelling-induced chloride current in Xenopus oocytes and have shown that a novel protein, pI/Cln, regulates this osmotically-sensitive chloride conductance. We have cloned pI/Cln, have shown that it is a ubiquous and highly conserved protein, and that it is required for the osmotic regulation of the chloride conductance. The goal of the current work is to 1) clone the osmotically-sensitive chloride conductance (I/Cl.swell) present in Xenopus oocytes and determine its interaction with pI/Cln; 2) identify other cytosolic and cytoskeletal-associated proteins binding pI/Cln; 3) determine functionally important properties of pI/Cln, in particular the effect of acidic domain-specific tyrosine phosphorylation of pI/Cln's subcellular localization, and changes that occur with shifts in osmolarity; 4) delete the I/Cln gene from embryonic stem cells and mice using established methods in our transgenic facility. Isolated embryonic stem cells and cells from knockout mice will be examined electrophysiologically for a hypotonic response.